Project Summary Red blood cell (RBC) storage under routine blood banking procedures may compromise RBC viability leading to altered rheological properties, loss of membrane integrity, and hemolysis. Cell-free hemoglobin may increase the risk of transfusion injuries, such as endothelial dysfunction and hypertension, via multiple mechanisms including interference with nitric oxide (NO) signaling, oxidative stress, and inflammation. We hypothesize that donor genetic predisposition to hemolysis may modulate the quality of RBC units, and the risk of transfusion injuries. In our collective studies, we find that male sex is significantly associated with increased levels of hemolysis in response to routine storage or in response to osmotic, mechanical or oxidative stress. Our gonadectomy studies in mice revealed that manipulation of testosterone levels via orchiectomy or testosterone repletion may modulate hemolytic response to osmotic or oxidative stress, whereas no such effect was observed with ovariectomy. Furthermore, our preliminary genome wide association studies in 22 inbred mouse strains suggested that sex differences in predisposition to hemolysis involve gene networks surrounding p38 MAPK, which may intensify hemolytic response during stress, and can be activated by testosterone. In this proposal, we more specifically hypothesize that exposure to testosterone during erythropoiesis promotes mitogen and stress-induced protein kinase activation leading to sex differences in erythrocyte biophysical properties, as well as to enhanced p38 MAPK activity under stress conditions, such as RBC storage and hemolytic disease. To test this hypothesis, we will verify whether orchiectomy in FVB/NJ mice correlates with improved RBC storage and transfusion outcomes compared with intact males, and whether testosterone repletion in orchiectomy mice compromises RBC recovery following storage and transfusion. We also propose to study the effect of testosterone repletion on RBC function in men undergoing testosterone therapy. Subsequently, genome wide association studies of gender differences and hemolytic propensity in 42 inbred mouse strains are set to identify gene networks that modulate hemolysis and may be regulated by testosterone signaling. To test the hypothesis that testosterone increases susceptibility to hemolysis via p38 MAPK signaling, we will monitor for changes in p38 MAPK expression during erythropoiesis of human hematopoietic stem cells treated with testosterone, in RBC membranes from patients undergoing testosterone treatments, and in-vivo using a mouse strain with attenuated p38 MAPK activity (B6.Cg-Mapk14tm1.1Dvb/J). We will characterize the p38 MAPK pro-hemolytic signaling cascade by studying its interactions with spleen tyrosine kinase (Syk) and Band 3 protein, as well as examine the therapeutic potential of MAPK and Syk inhibitors to improve the recovery and quality of stored RBCs. Taken together, this proposal offers novel mechanistic understanding of sex differences in hemolysis highly relevant to the prevention of transfusion-related injuries.